Nucleic acid amplification techniques and assays are well known. Some reactions for amplifying DNA are isothermal, such as nucleic acid sequence base amplification (NASBA). Others employ thermal cycling, such as the polymerase chain reaction (PCR). Amplification and assays employing amplification utilizing PCR are described, for example, in U.S. Pat. Nos. 4,683,202, 4,683,195, and 4,965,188, and, generally, PCR PROTOCOLS, a guide to Methods and Applications, INNIS et al. eds., Academic Press (San Diego, Calif. (USA) 1990), each of which is hereby incorporated by reference in its entirety. PCR amplifications are generally designed to be symmetric, that is, to make double-stranded products (or “amplicons”) by utilizing equimolar or approximately equimolar concentrations of a pair of matched primers, that is, a forward primer and a reverse primer that have equal melting temperatures (Tm's). A technique that has found limited use for making largely single-stranded amplicons directly in a PCR amplification reaction is “asymmetric PCR,” described in Gyllensten and Erlich, “Generation of Single-Stranded DNA by the Polymerase Chain Reaction and Its Application to Direct Sequencing of the HLA-DQA Locus,” Proc. Natl. Acad. Sci. (USA) 85: 7652-7656 (1988); and U.S. Pat. No. 5,066,584. Asymmetric PCR is a non-symmetric PCR amplification method that differs from symmetric PCR in that one of the primers is diluted fivefold to one hundredfold so as to be present in limiting amount of 1-20 percent of the concentration of thee other primer. As a consequence, the amplification consists of an exponential phase in which both primers are extended, generating double-stranded amplicon, followed by linear amplification in which only one primer remains, generating single-stranded amplicon.
A more recent non-symmetric PCR amplification method is “Linear-After-The-Exponential PCR” LATE-PCR), which utilizes primers in different concentrations but wherein the primers are not “matched” as in symmetric PCR and asymmetric PCR. Sanchez et al. (2004) Proc. Natl. Acad. Sci. (USA) 101:1933-1938, published international patent application WO 03/054233 (3 Jul. 2003), and Pierce et al. (2005) Proc. Natl. Acad. Sci. (USA) 102; 8609-8614, all of which are incorporated herein by reference in their entirety. DNA amplification methods can be used for RNA targets by first performing reverse transcription to create cDNA, which is then amplified, for example, by one of the foregoing PCR methods.
Detection and analysis of nucleic acid amplification products can be performed in a variety of ways. Double-stranded amplicons can be monitored with a dye that fluoresces upon intercalating into or otherwise interacting with double-stranded DNA, such and SYBR Green or SYBR Gold. See, for example, U.S. Pat. No. 5,994,056. Amplicons can be subjected to a sequencing reaction, for example, conventional dideoxy sequencing or Pyrosequencing, a real-time sequencing-by-synthesis method. Hybridization probes are commonly used for detection. Probes may be labeled or unlabeled. Detection of hybridized probes may be by a physical characteristic, such as size, by participation in a subsequent event, for example, a color-forming reaction, or by detection of a label applied to the probe, such as a radioactive or fluorescent label. Examples of labeled probes are 5′ Nuclease probes that are cleaved during primer extension (U.S. Pat. Nos. 5,210,015, 5,487,972 and 5,538,848), molecular beacon probes (U.S. Pat. Nos. 5,925,517, 6,103,476 and 6,365,729), Yin-Yang double-stranded probes (Li, Q. et al (2002) Nucl. Acids Res. 30:e5) and FRET probe pairs.
All of the above PCR based methods of amplification depend on the action of a thermostable DNA polymerase recovered from bacterial source. In their native form these enzymes are single polypeptides with several domains and several activities: including a 5′ to 3′ polymerase, a 5′ to 3′ exonuclease, and a 3′ to 5′ editing function (which is deleted from commercially used enzymes). Taq DNA polymerase (from Thermus aquaticus) is the most widely used, including hot-start forms, but Tfi DNA Polymerase (Invitrogen, Inc, product #30342-011) is another such enzyme. In addition to these thermostable DNA polymerases there are several thermostable DNA polymerases which also carry out reverse transcription of RNA into DNA, along with polymerization of DNA strands and exonuclease cleavage of certain 5′ ends. These enzymes include ZO5 polymerase and Thermus thermophilus (TTH) polymerase.
The 5′ to 3′ exonuclease activity found in the thermostable DNA polymerases has been much studied using Taq polymerase. For instance, this exonuclease activity is the basis of so-called 5′ nuclease assays used in connection with symmetric PCR. The 5′ nuclease assays utilize two primers and a probe. The probe is a linear, or random coil, DNA oligonucleotide having a fluorophore covalently linked to one terminal nucleotide and a nonfluorescent quencher covalently linked to the other terminal nucleotide. It hybridizes to one of the two target strands to which one of the two primers binds. The melting temperature of a 5′ nuclease probe is higher than that of its upstream primer, and the probe is therefore located downstream of the extending primer. The 5′ to 3′ exonuclease activity of the Taq polymerase encounters and cleaves the 5′ end of the probe as the 5′ to 3′ polymerase domain of the enzyme extends the 3′ end of the primer. If the probe has a fluorescent moiety on its 5′ end, that moiety and the nucleotide to which it is covalently linked are separated from the rest of the oligomer by cleavage. If the remainder of the oligonucleotide is still bound to the target sequence, it is cleaved again by the 5′ exonuclease of the advancing polymerase. This is primer-dependent cleavage of the probe.
Primer-dependent cleavage of the probe has the following characteristics: 1) The 3′ end of the primer must have an unblocked (or uncapped) 3′ —OH group. Thus, addition of —PO4 or other chemical moiety to the 3′ —OH, or removal of the 3′ OH, prevents cleavage. 2) The primer must advance up to and/or “invade under” the 5′ end of the probe. Thus, except as noted below, omission of one or more nucleotide triphosphates from a primer-dependent reaction will prevent cleavage, if the primer cannot advance up to the 5′ end of the probe. The exception to this rule is that a primer with a 3′ OH can be designed which already invades under the 5′ end of the probe without additional extension. 3) If the 3′ end of the primer that already invades under the 5′ end of the probe, that 3′ end must be complementary to the target sequence. Thus, a non-complementary 3′ extension (arm) of 2 or more nucleotides at the 3′ end of the primer prevents primer dependent cleavage of the probe, even if the 3′ —OH is uncapped. Removal of the 5′ to 3′ domain of Taq polymerase generates an enzyme known as the Stoffel fragment. PCR amplifications utilizing the Stoffel fragment cannot use 5′ nuclease (TAQMAN, a trademark of Roche Molecular Systems)) probes. Construction of probes, such as molecular beacons, using certain modified nucleotides, such as 2′ o-methyl nucleotides across their entire length prevent primer-dependent cleavage the probe.
Lyamichev et al. (Biochemistry (2000) 39: 9523-9532) described an invasive signal amplification reaction based on the three oligonucleotide structural features characteristic of primer-dependent cleavage of a probe. They reported that “by running the reaction at an elevated temperature, the downstream oligonucleotide cycles on and off the target leading to multiple cleavage events per target molecule without temperature cycling”.
The 5′ to 3′ exonuclease activity of thermostable DNA polymerases is also known to carry out primer-independent cleavage of a probe-target hybrid. This reaction has been studied using hairpin-shaped target molecules with stems of 16 base-pairs, loops of 4 nucleotides, and 5′ ends that are labeled with P32—PO4. The two arms of the hairpin are either equally long (i.e. it is blunt ended), or the 3′ arm extends beyond the 5′ end, or the 5′ end extends beyond the 3′ end. Using molecules of this design the primer-independent 5′ to 3′ exonuclease activity of Taq Polymerase has been shown to have the following characteristics: 1) Lyamichev, V., et al. (Science 260, 778-783 (1993)) reported that in the absence of a primer the 5′ to 3′ nuclease of Taq polymerase cleaved the recessed 5′ end of a hairpin substrate between the last two base pairs of the substrate strand and the target strand. 2) Lyamichev et al. (Proc. National Acad. Sci. 96: 6143-6148 (1999)) demonstrated that the 5′ to 3′ exonuclease activity of intact Taq polymerase (TaqNP) does not efficiently cleave the 5′ end of a hairpin structure whose 3′ end was recessed by 6 nucleotides. These authors concluded that “low efficiency of cleavage probably results from binding of the polymerase domain of this enzyme to the end of the duplex, which resembles a template-primer complex”. They did not test subtrates that resemble probes hybridized to targets rather than primers hybridized to targets.
For certain amplification objectives and for certain detection objectives, the amplification methods and the detection methods known in the art are unsuitable or have limitations. For example, multiplexed PCR assays can only distinguish among five or six targets by differently colored fluorescent probes. Also, it is very difficult to detect a rare allele in a sample containing an abundant allele.